1. Field of Invention
The present invention relates to Kex2 derivatives with Kex2 protease activity which are secreted in large amount in culture medium, and to a method for their production. The invention also relates to a method of using the aforementioned secretory Kex2 derivatives.
2. Related Art
Many attempts have been made at methods for producing physiologically active peptides by chimeric protein expression, and chemical or enzymatic cleavage methods have been used for release of the desired proteins. Chemical methods include cleavage of asparagine residue with nitrous acid and cleavage of methionine residue with CNBr (Itakura et al., Science 198, 1059, 1977). However, these methods necessarily involve modification of the protein of interest, and problems of purification cost.
Enzymatic methods employ lysyl endopeptidase which specifically cleaves the peptide bond of the C-terminal of lysine (Achromobacter protease I) and Staphylococcal protease V8 which specifically cleaves the peptide bond of the C-terminal of the glutamic acid (Japanese Examined Patent Publication No. 6-87788). However, since these chemical methods and endoproteases recognize a single amino acid residue, it is a precondition that amino acid residue not be present in the desired peptide in order to allow efficient excision of the desired peptide from the chimeric protein, and thus the peptides which can be produced are limited. Efforts have therefore been directed at developing a highly universal cleavage method which recognizes multiple amino acid residues.
Prohormone converting enzymes are enzymes which produce peptide hormones from their precursors in vivo, and they are expected to have favorable qualities as enzymes for excision of peptide hormones from proteins, even in vitro. Kex2 protease is a prohormone converting enzyme derived from Saccharomyces cerevisiae, and it is a calcium-dependent serine protease which specifically cleaves peptide bonds at the C-terminal ends of Lys-Arg, Arg-Arg and Pro-Arg sequences. Kex2 protease is a protein composed of 814 amino acid residues with a signal sequence at the N-terminus and a transmembrane region at the C-terminus with a continuous string of hydrophobic amino acids, and it is localized in the trans Golgi in cells.
A nucleotide sequence coding for Kex2 protease and the corresponding amino acid sequence are shown in the Sequence Listing as SEQ ID NO.1. Genetic expression of a Kex2 derivative lacking the C-terminal region in Saccharomyces cerevisiae and subsequent analysis thereof revealed that the Kex2 derivative with the amino acid sequence from amino acids 1 to 614 of SEQ ID NO.1 retains the Kex2 protease activity, and is secreted in culture medium (Fuller et al., Proc. Natl. Acad. Sci. USA, 86, 1434-1438, 1989, Japanese Unexamined Patent Publication No. 1-199578). In the present specification, the Kex2 protease derivative is represented by the number of amino acids counting from amino acid 1 of SEQ ID NO.1. For example, the Kex2 derivative with the amino acid sequence from amino acids 1 to 614 of SEQ ID NO.1 is represented as Kex2-614.
Heretofore known Kex2 derivatives whose secretory production methods have been studied include ss-Kex2 and Kex2.DELTA.p.
ss-Kex2 is a Kex2 derivative which has a 3 amino acid residue peptide added to Kex2-614, and its production in Saccharomyces cerevisiae has been studied (Brenner et al., Proc. Natl. Acad. Sci. USA, 89, 922-926, 1992). It was expressed in a protease-deficient mutant (pep4) as a host (in a 4 mg/L culture medium), and was purified from the culture supernatant at a purification yield of 20%. The reduced molecular weight of the purified ss-Kex2 treatment with Asn-type sugar chain hydrolyzing enzyme EndoH suggests that it includes Asn-type sugar chains. The pH dependency and substrate specificity of the enzyme activity has also been studied using synthetic substrates.
Kex2.DELTA.p is a Kex2 derivative represented in this specification by Kex2-666, and studies of its production in the insect cell host Sf9 have shown that 90% of its activity is secreted into the culture supernatant, and that the molecular weight of the secreted Kex2.DELTA.p is 70 kDa, which is smaller than the intracellular molecular weight of 120 kDa (Germain et al., Eur. J. Biochem. 204, 121-126, 1992). In addition, since the 70 kDa molecular weight protein is found in the culture supernatant in which Kex2 is expressed, and replacement of the 385th serine residue by alanine residue of Kex2.DELTA.p (the catalytic portion of Kex2 protease activity) results in Kex2.DELTA.p in the culture supernatant with a molecular weight of 120 kDa, equal to the intracellular molecular weight, the 70 kDa protein is believed to be an autolysate of the C-terminal portion-deficient Kex2.DELTA.p (120 kDa) in the culture medium.
Attempts have also been made at expression of the derivative Kex2.DELTA.504 in which the cleavage site of the Lys-Arg sequence (amino acids 503-504 of SEQ ID NO.1), expected from the molecular weight of the decomposition product and the substrate specificity of Kex2 protease, is replaced with the Lys-Leu sequence. However in this case as well a 70 kDa protein is found in the culture medium, and since the Lys-Arg sequence is not always cleaved by Kex2.DELTA.p during autolysis, and no other sequence exists as the recognition site of Kex2 protease, this suggests the possibility that Kex2.DELTA.504 recognizes a completely different sequence than the one predicted from the synthetic substrate, and cleaves itself.
Thus, despite research on substrate specificity of Kex2 derivatives using synthetic substrates, the substrate specificity when using proteins is not yet understood. Also, little is known about the secreted amounts of the different Kex2 derivatives, and it is still not known whether stable secretory production of Kex2 derivatives other than Kex2-614 is possible.